The long-term goal of this project is to understand how primary sequence directs the expression of eukaryotic genes and the structure and function of their encoded protein products. The human gene encoding galactose-1- phosphate uridyltransferase (GALT), an essential enzyme of the Leloir pathway of galactose metabolism, will be used is a model for study. Mutations in human GALT result in the potentially lethal disorder galactosemia, which is inherited as an autosomal recessive trait. Approximately 1/30,000 to 1/60,000 live-born infants suffer from severe, or "classic" galactosemia, while about 1/11,000 infants exhibit milder, or "variant" forms of this disease. Both the cDNA and gene encoding human GALT have recently been cloned, paving the way for molecular studies of the gene, the enzyme, and the disease. The specific objectives of this proposal are to identify and characterize essential functional and regulatory elements within human GALT. The Specific Aims therefore are: (1) to analyze the molecular and biochemical effect(s) of known naturally occurring GALT mutations derived from patients with galactosemia, (2) to perform a directed mutational analysis of selected domains of human GALT, and (3) to clone and characterize the human GALT promoter. Naturally-occurring mutations in human GALT have been and will continue to be identified by direct-sequencing of PCR-amplified GALT cDNA and genomic sequences derived from galactosemia patients and their relatives. Site- specific mutations will be generated by oligonucleotide-directed mutagenesis of the cloned wildtype sequence. The effects of both naturally-occurring and directed mutations within the GALT coding region will be assessed using a yeast-based expression system for the human enzyme. This yeast-based system has already been established and shown to facilitate both genetic and biochemical manipulations of the human cDNA and enzyme. The human GALT promoter will be cloned by inverse or one-sided PCR extending from known sequences near the 5' end of the gene. Once cloned, the promoter will be characterized functionally by deletion analysis and mammalian cell transfection in the context of an established mammalian cell reporter gene construct. Combined, these studies should provide insights that are meaningful not only in terms of the basic science of eukaryotic gene expression and structure/function relationships, but also in terms of advancing the diagnosis, prognosis, and treatment of patients with galactosemia.